*2.3. Reverse Phase-Liquid Chromatography Mass Spectrometry Analysis (RP-LC-MS*/*MS)*

The digested protein samples (above) were resuspended in 10 µL of 0.1% formic acid and analyzed by RP-LC-MS/MS in an Easy-nLC II system coupled to an ion trap LTQ-Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Fisher Scientific). The peptides were concentrated (on-line) by reverse phase chromatography using a 0.1 × 20 mm C18 RP precolumn (Thermo Fisher Scientific) and then separated using a 0.075 × 250 mm C18 RP column (Thermo Fisher Scientific) operating at 0.3 µL/min. Peptides were eluted using a 180-min dual gradient. The gradient profile was set as follows: 5−25% solvent B for 135 min, 25−40% solvent B for 45 min, 40−100% solvent B for 2 min, and 100% solvent B for 18 min (Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid and 80% acetonitrile in water). ElectroSpray ionization (ESI) was done using a nano-bore emitter stainless steel ID 30 µm (Proxeon) interface. The Orbitrap resolution was set at 30,000. Peptides were detected in survey scans from 400 to 1600 amu (1 µscan), followed by twenty data-dependent MS/MS scans (Top 20) using an isolation width of 2 u (in mass-to-charge ratio units), a normalized collision energy of 35%, and a dynamic exclusion that was applied during 60 s periods.
